Improving the thiobarbituric acid-reactive-substances assay for estimatinglipid peroxidation in plant tissues containing anthocyanin and other interfering compounds

The occurrence of malondialdehyde (MDA), a secondary end product of the oxi dation of polyunsaturated fatty acids, is considered a useful index of gene ral lipid peroxidation. A common method for measuring MDA, referred to as t he thiobarbituric acid-reactive-substances (TBARS) assay, is to react it wi th thiobarbituric acid (TBA and record the absorbance at 532 nm. However, m any plants contain interfering compounds that also absorb at 532 nm, leadin g to overestimation of MDA values. Extracts of plant tissues including purp le eggplant (Solanum? Melongena L.) fruit, carrot (Daucus carota L.) roots, and spinach (Spinacia oleracea L.) leaves were assessed for the presence o f MDA and other non-MDA compounds absorbing at 532 nm. A method described h erein corrects for these interferences by subtracting the absorbance at 532 nm of a solution containing plant extract incubated without TEA from an id entical solution containing TEA. The reliability and efficiency of this spe ctrophotometric method was assessed by altering the relative ratios of exog enous MDA additions and/or extracts of red cabbage (Brassica oleracea L.) l eaves containing interfering compounds and then measuring MDA recovery. Rel iability was also validated through high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry techniques. Re sults indicated that over 90% of exogenously added MDA could be recovered t hrough the improved protocol. If there were no corrections for interfering compounds, MDA equivalents were overestimated by up to 96.5%. Interfering c ompounds were not detected in vegetables such as lettuce (Lactuca sativa L. ) and spinach which had low or negligible concentrations of anthocyanidin d erivatives. Comparisons between the TEARS method presented here and two cur rently accepted protocols indicated that the new modified method exhibits g reater accuracy for quantifying TBA-MDA levels in tissues containing anthoc yanins and/or other interfering compounds. This modified protocol represent s a facile and rapid method for assessment of lipid peroxidation in virtual ly all plant species that contain interfering compounds.